Growth factor IGF-Il

ABSTRACT

The present invention relates to the use of iGF-II or effective analogues thereof for the manufacture of a medicament for prevention or treatment of nutritional or gastrointestinal disorders and for promoting human or animal neonatal growth. It also relates to composition comprising exogenous human or animal IGF-II or effective analogues thereof in a therapeutically effective amount together with a pharmaceutically acceptable carrier or diluent or foodstuff, preferably in admixture with artificial or natural milk or colostrum. The invention may be applied both in man and in animals.

The present invention; relates to the use of IGF-II or effectiveanalogues thereof for the manufacture of a medicament for prevention ortreatment of nutritional or gastrointestinal disorders and for promotinghuman or animal neonatal growth. It also relates to compositioncomprising exogenous human or animal IGF-II or effective analoguesthereof in a therapeutically effective amount together with apharmaceutically acceptable carrier or diluent or foodstuff, preferablyin admixture with artificial or natural milk or colostrum. The inventionmay be applied both in man and in animals.

INTRODUCTION AND PRIOR ART

Insulin-like growth factor 2 (IGF-II) is a peptide present in plasma andother body fluids. Its primary sequences shows 64% homology to IGF-I andcomprises 67 amino acids, including 3 disulphide bonds. It can stimulategrowth of a wide range of cell types. IGF-II have been purified fromhuman plasma and the complete amino acid sequence is known. Sequenceswith extensive homologies to human IGF-II are present in IGF-II purifiedfrom plasma of other species. IGF-II has systemic and local effects andappear mostly associated with different specific binding problems, sixof which are sequenced and are termed IGFBP1, IGFBP2, IGFBF3, IGFBP4,IGFBP5 and IGFBP6. These appear to modulate the biological functions andavailability of IGF-II in both a positive and negative manner. IGF-IIappears to act mainly by interactions with the IGF-type 1 receptorexposed on the outer surface of plasma membranes in many different celltypes--however relative specificity of action rosy be found because ofthe influence of binding proteins. IGF-II may also have distinct actionsas it binds to a distinct and unrelated type 2 receptor also found oncell membranes. Moreover, binding of IGF-II to insulin receptors alsoseems to be of importance. Because of the scarcity of purified plasmaIGF-II there was a great necessity to develop methodology for thecommercial scale production of IGF-II. Nowadays such large scaleproduction can readily be achieved by using recombinant DNA techniques.IGF-II has been shown to experimentally reduce the catabolic state instarved animals and to antagonise some metabolic actions of IGF-I (Koeaet al Endocrinology 1992, 130, 2423-2425). These observations of Koea etal plus the different range of receptor specificities of IGF-II to thatof IGF-I mean that there is no obviousness to any action of IGF-II onthe gastrointestinal tract.

It has previously been demonstrated that both type I and type 2 IGFreceptors are present in the gastrointestinal tract and that oral IGF-Iand IGF-II affect jejunal enzymes following repeated administration inolder suckling rats, but no effect on intestinal growth was observed.(Young et al. Digestion 46, (1990), Suppl. 2, 240-252). Ballard et al ,WO 91/12018 have disclosed the therapeutic use of IGF-I forgastrointestinal disease or the treatment of the shortened gut aftersurgery. Ballard provide no evidence of activity following oraladministration.

Heinze-Erian et al, Endocrinology, (1991) Vol 129, No 4, 1769, reportsthat there is an essential role for both IGF receptors in the regulationof cell mitogenesis and growth.

Grey Vet al, Mol-Cell-Endocrinol, 1991, Mar, Vol 75 (3), 221-7 suggeststhat IGF-II/man-6-P receptor may play a role in the adaptiveregenerative response of the intestinal epithelium.

There was however not a priori reason to believe that IGF-II might beefficacious in the normal gut in the premature or immediately neonatalgut and the prior art does not demonstrate any known action of IGF-II onthe gastrointestinal system.

No studies of the effects of oral administration of the IGF-II on theneonatal gastrointestinal system, nutritional status or growth have beenpreviously suggested.

There is a need for a medicament for prevention or treatment ofnutritional or gastrointestinal disorders.

It has now surprisingly been found that IGF-II or effective analoguesthereof can be used for the manufacture of such a medicament which alsocan be used for promoting human or animal neonatal growth.

THE INVENTION

The invention relates to the use of IGF-II or effective analoguesthereof for the manufacture of a medicament for prevention or treatmentof nutritional or gastrointestinal disorders and for promoting human oranimal neonatal growth.

The medicament promotes e.g. intestinal mucosal growth and cellproliferation, intestinal and gastointestinal muscle growth followingsurgery or in disorders of the gastrointestinal tract.

The medicament can restore or maintain gastrointestinal function afterperiods of parenteral nutrition or after gastrointestinal diseaseincluding gastroenteritis, inflammatory bowel disease, bowel surgeryulceration of the duodenum or to accelerate recovery.

The medicament also promotes villous growth and enhances nutritionalstatus in patients with mucosal villous diseases such as coeliacdisease, post infective villous atrophy and short gut syndromes.

Another aspects of the invention is the use in which the medicamentpromotes growth of neonatal and premature human or animal, such asgrowth of premature infants, reduces the risk of necrotisingenterocolitis and reduces the risk of infection of the gastrointestinaltract.

The medicament promotes villous growth and enhance nutritional status inpremature infants or in growth retarded infants and reduces the risk ofenteric disease such as enteritis in the neonate.

Human or animal IGF-I can be used and may be given singly or incombination with other growth factors such as IGF-I and epidermal growthfactor (EGF) for enhancing or improving the desired effect(s) of IGF-IIor its effective analogues.

The invention also relates to a composition comprising human or animalIGF-II or effective analogues thereof in a therapeutical effectiveamount together with a pharmaceutically acceptable carrier or diluent,preferably adapted for oral, gastric or interna; administration and mostpreferably together with milk or colostrum.

This composition is intended for promoting growth, thrift or nutritionin man or animals or for reducing the risk of intestinal infection.

A Regarding the use for prevention or treatment of nutritional orgastrointestinal disorders, the following uses are pointed out:

I In man:

1) Promotion of villous growth in older subjects In diseases associatedwith villous atrophy, flattening or where there is malabsorptionincluding coeliac disease, cystic fibrosis, inflammatory diseases of thebowel (e.g. Crohns disease), following gastroenteritis, followingintestinal surgery including resection.

2) Treatment of inflammatory diseases of the bowel (e.g. Crohnsdisease).

3) Treatment of post-inflammatory villous atrophy.

4) Treatment of malabsorption disorders.

5) Treatment of short gut syndromes.

6) Acceleration of gut recovery following bacterial,viral or amoebicenteritis or giardiasis.

7) Prophylaxis against necrotising enterocolitis.

8) Maintenance of gastrointestinal function in parenterally fedsubjects, particularly neonates.

9) Acceleration of growth in growth retarded infants.

II In animals:

1) Prophylaxis or treatment of inflammatory conditions of thegastrointestinal tract particularly in infancy.

2) Promotion of infant growth by supplementation of artificial ornatural milks fed to infant animals.

3) Treatment of gastrointestinal disorders or disturbances at weaning.

4) Promotion of growth of the smooth muscle layer of the intestine. Insituations such as following intestinal surgery full gastronutritionalfunction may be recovered more quickly if muscular growth is enhanced.

In primary constipation, or secondary to disorders of neural innervationof the gastrointestinal system increased muscle mass may be of value.

In animal either recombinant human IGF-II or IGF-II of other species(e.g. bovine, porcine) may be used either as an oral drench or as asupplement to artificial liquid or solid feeds.

B. Regarding the use for promoting human or animal neonatal growth thefollowing uses are pointed out, but are not limited to:

1) Promotion of intestinal mucosal cell proliferation and villous growthand/or crypt depth in the neonate.

The property of IGF-II or its effective analogues when administeredorally in the neonate is useful for the prevention treatment of avariety of primary or secondary neonatal nutritional andgastrointestinal disorders and in improving the management of theneonate.

2) Promotion of growth, gut and pancreatic development in growthretarded newborns.

3) Acceleration of the initiation of oral feeding in premature infants.

4) Reducing the risk of necrotising enterocolitis in the prematureinfant.

5) Improving nutritional status in the growth retarded infant.

6) Preservation of gastrointestinal function and growth in infantsdeprived of oral feeding for prolonged periods (for example extremeprematurity or following gastrointestinal surgery) or acceleration ofswitching from parentoral to oral feeding in such infants.

7) As a supplement to the management of the neonates, infants orchildren with deficient growth or health associated with impaired oralnutrition for example following gastrointestinal infection or associatedwith specific disease of the gastointestinal tract such as followingnecrotising enterocolitis, following gastrointestinal surgery or withprimary or secondary villous atrophy.

8) Acceleration of normal infant growth in animal.

Preferably in man human IGF-II singly or in combination with IGF-I isused. The dose given could be 0.01 to 100 μg/kg/body weight per day. Thepreferred route of administration is by mouth either in aqueous bufferor other pharmacological composition or added to artificial feed,artificial or natural milk. Alternatively it may be installed moredistally in the gastrointestinal tract for example by nasogastric tubeor by duodenal tube.

C Pharmaceutical composition comprising IGF-II.

The present invention in respect of all the above provides apharmaceutical composition that includes an effective amount of apeptide or effective analogue of IGF-II and a pharmaceuticallyacceptable carrier or diluent, preferably adapted for oral, gastric, orinternal administration. The peptide may be present in amountssufficient to provide a dose rate of approximately 0.01 to 100 μg/kgbody weight per day, preferably 0.1-10 μg/kg per day.

D. The claimed composition can be used as a supplement to foodstuff,

The foodstuff is preferably milk to promote nutrition, growth and reducethe impact of gastrointestinal infection. It is desirable in humaninfants with growth failure, prematurity of where there is difficulty inestablishing oral feeding. The use is particularly desirable in infantanimals from large litters, on artificial feeds or where there is growthretardation present. The invention also extends to a nutritionallyacceptable composition for the supplementation of natural or artificialmilk formula for human or animal use such that IGF-II or analogue isprovided. The peptide may be present in amounts sufficient to provide adose rate of approximately 0.01 to 100 μg/kg body weight per day,preferably 0.1-10 μg/kg per day.

DETAILED DESCRIPTION OF THE INVENTION

The preferred form of the invention will now be described with referenceto the following non-limiting example.

EXAMPLE 1

Studies were performed in newborn piglets that were raised for 24 hoursfollowing birth with a commercial infant milk formula (SMA Gold Cap;John Wyeth & Bro (NZ) Ltd) containing undetectable (<1 ng/ml) levels ofIGF-I or IGF-II or with the same formula supplemented with either 2μg/ml of recombinant human IGF-I or recombinant human IGF-II (providedby Kabi Pharmacia AB, Sweden). 7 piglets received each treatment. Thepiglets were from 7 litters and each litter provided on one formula fedand one formula plus IGF-II fed piglet. The piglets had statisticallysimilar birth weights. After birth the piglets were fed by bottle 20ml/kg every 2 hours for the first 12 hours, then 40 ml/kg every 4 hoursthereafter until slaughter. The animals were slaughtered at 24 hoursafter birth for histological evaluation.

In addition the rate of cell proliferation was assessed by administeringbromodoxyuridine (BRDU) intraperitoneally to the piglet in 4 equal dosesof 5 mg/kg at 30 minute intervals between 120 and 30 mins prior toslaughter. BRDU labelling can be detected histologically and indicatesactive cell proliferation. The net weights of the cleanedgastrointestinal tract components were compared (see Tables 1 to 3).Tissue blocks were taken from the mid duodenum, the proximal and distaljejunum and the proximal and distal ileum and histological measurementswere made using images projected onto a digitizer pad and quantified bycomputer programme (Sigma Scan). Further analysis for RNA, DNA andprotein content were performed on proximal jejunal mucosa (Table 4).

                  TABLE 1                                                         ______________________________________                                        Mean body-weight and weights and physical dimensions of                       digestive organs in 24 hour old piglets raised on an infant formula           with or without addition of IGF-II.                                                             Treatment                                                                     Control                                                                              IGF-II                                                                 n = 7  n = 7                                                ______________________________________                                        Birth Weight (kg)   1.286    1.295                                            Final Weight (kg)   1.318    1.320                                            Stomach weight (g/kg)#                                                                            5.02     4.99                                             Pancreas weight (g/kg)#                                                                           1.23     1.37*                                            Small intestine:                                                              Weight (g/kg)#      29       29                                               Length (cm/kg)#     310      323                                              Mitotic index       6.93     8.63***                                          (cells/crypt labelled)                                                        Large intestine:                                                              Weight (g/kg)#      6.2      6.4                                              Length (cm/kg)#     69       72                                               ______________________________________                                         #Adjusted for the birth weight.                                               *p < 0.05?                                                                    ***p < 0.001                                                             

                  TABLE 2                                                         ______________________________________                                        Effects of oral ingestion of IGF-II on the mean weights of small              intestinal mucosal and muscle (n = 5/group).                                                      % difference from                                                      Control                                                                              control                                                                (g)    IGF-II                                                    ______________________________________                                        Duodenal mucosa                                                                              0.54     15.6                                                  Duodenal muscle                                                                              0.44     19.4                                                  Jejunal mucosa 9.90     30.2                                                  Jejunal muscle 3.27     18.7                                                  Ileal mucosa   7.82     29.2                                                  Ileal muscle   3.15     36.3                                                  ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Mean microscopic measurements (μm) and mean cell prolif-                   eration rate in the small intestine of 24 hour old piglets                    raised on an infant formula with or without addition of                       IGF-II (n = 5/group).                                                                         Control                                                                              IGF-II                                                 ______________________________________                                        Wall thickness    1019     1100                                               Villus length     725      784                                                Crypt depth       97       106                                                Submucosa thickness                                                                             93        94                                                Muscularis thickness                                                                            79        88                                                ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Chemical compositions of the proximal jejunal mucosa.                         (n = 5/group)                                                                                   Control                                                                              IGF-II                                               ______________________________________                                        Weight (g/kg)#      5.712    6.147                                            Protein (mg/kg)#    583      559                                              RNA (mg/kg)#        28.9     31.0                                             DNA (mg/kg)#        25.0     28.1                                             Protein: DNA        23.0     20.0                                             RNA: DNA ratio (mg/mg)                                                                            1.14     1.11                                             ______________________________________                                         #Adjusted for the birth weight.                                          

These observations provide clear evidence that in neonatal animals oraladministration of IGF-I is selectively active to promote mitosis ofcrypt cells in the small intestine. These cells are the source of theenterocytes that form the absorptive layer on the intestinal villous. Itwill also promote intestinal growth of the villous mucosa and promotemuscle growth.

Finally it has to be understood that various other modifications and/oralterations may be made without departing from the spirit of the presentinvention as outlined therein.

We claim:
 1. Composition for the treatment of nutritional orgastrointestinal disorders or for promoting human or animal neonatalgrowth comprising human or animal IGF-II in a therapeutical effectiveamount together with a pharmaceutically acceptable carrier or diluent,wherein said carrier or diluent is for oral or gastric administrationand wherein said carrier or diluent comprises natural milk orcolorstrum.
 2. The composition of claim 1 Wherein said carrier ordiluent is for oral administration.
 3. Composition according to claim 1for promoting growth, thrift or nutrition in man or animals or reducingthe risk of intestinal infection.
 4. The method for the treatment ofnutritional or gastrointestinal disorders or for promoting human oranimal neonatal growth in a patient in need thereof which comprisesadministering to said patient a medicament for oral or gastricadministration and wherein said carrier or diluent comprises naturalmilk or colostrum comprising a therapeutically effective amount ofIGF-II.
 5. The method of claim 4 wherein said IGF-II is human IGF-II. 6.The method of claim 4 wherein said IGF-II is animal IGF-II.
 7. Themethod of claim 4 wherein said medicament is administered orally.
 8. Themethod of claim 4 for promoting intestinal mucosal growth.
 9. The methodof claim 4 for promoting gastrointestinal muscle growth followingsurgery or in disorders of the gastrointestinal tract.
 10. The method ofclaim 4 for restoring or maintaining gastrointestinal function afterperiods of parenteral nutrition or after gastrointestinal disease or toaccelerate recovery.
 11. The method of claim 4 for promoting villusgrowth and enhances nutritional status in patients with mucosal villousdiseases.
 12. The method of claim 4 for promoting growth of neonatal andpremature human or animal.
 13. The method of claim 12 for promotinggrowth and development of neonatal animals, growth of premature infantsand reducing the risk of necrotizing enterocolitis and infection ofgastrointestinal tract.
 14. The method of claim 12 for promoting villousgrowth and enhanced nutritional status in premature infants or in growthretarded infants.
 15. The method of claim 12 for reducing the risk ofenteric disease.
 16. The method of claim 10 wherein said disease isselected from the group consisting of gastroenteritis, inflammatorybowel disease, and bowel surgery ulceration of the duodenum.
 17. Themethod of claim 11 wherein said mucosal villous diseases are selectedfrom the group consisting of coeliac disease, past infective villousatrophy and short gut syndromes.
 18. The method of claim 12 for reducingthe risk of enteritis in a neonate.
 19. The method of claim 4 whereinsaid patient is a neonate.
 20. The method of claim 4 wherein saidpatient is a premature infant.
 21. The composition of claim 1 whichconsists of said IGF-II as the active ingredient.
 22. The method ofclaim 4 which consists of administering said IGF-II as the activeingredient.